Over the last decades, many biologically active compounds have been developed for the treatment of numerous diseases such as cancer, respiratory and metabolic diseases. Despite the great progress on the way towards the design, specificity and overall development of biologically active compounds, there are still serious issues such as poor bioavailability, safety and limited tissue distribution that hamper their preclinical and clinical applicability. The latter need to be circumvented in order some of these compounds can be safely and successfully applied in the research and clinical field.
Nucleic acid molecules are one major sub class of biologically active compounds and through the progress of the last 30 years, their use in the therapeutic field has evolved from basic science towards applied molecular therapy. Short nucleic acids, such as antisense oligonucleotides, ribozymes, microRNAs, decoys and small interfering RNAs, or long nucleic acids such as plasmids have the ability to regulate RNA. Therefore, the ability to regulate the expression of the target protein in a specific manner, offers unlimited potential for gene therapy, antisense therapy and RNAi therapy among others (Whitehead et al., (2009) Nature Review Drug Discovery 8:129-138). Still, tissue distribution, efficient uptake by the target cell and their trafficking into the cytosol are of major importance for the sequence specific gene regulation. As nucleic acids are large and negatively charged molecules, their passive diffusion through the negatively charged lipophilic cell membrane or their cytosolic internalization by the mechanisms of endocytosis is poor and limits their efficiency. Therefore, the assisted delivery of these nucleic acid molecules is desirable for successful research and therapeutic applications (Behlke, (2006) Molecular Therapy 13:644-670); de Foungerolles et al., (2007) Nature Review Drug Discovery 6:443-453).
Lipid assemblies including liposomes and lipoplexes are one common strategy among non-viral vectors for performing carriage of pharmaceutical substances to target cells. Thus, lipid assemblies have attracted substantial interest as delivery technologies for nucleic acids. In general, there are three main sub types of lipid particles, which have been used over the last decades as delivery systems. Depending on the biophysical properties and more specifically on the surface charge of the lipid membrane, lipid particles are divided into the following main categories: neutral, anionic and cationic lipid particles.
In the past years, only a few neutral and anionic liposomal vectors have been developed. These types of liposomal vehicles are prepared using either neutral lipids, or a combination thereof with anionic lipids. Due to the neutral or anionic charge of the bilayer, these types of lipid membranes demonstrate very low toxicity levels and exhibit relatively long circulation lifetimes, which increases nucleic acid tissue distribution (Landen et al., (2005) Cancer Res. 65:6910-6918 and Halder et. al., (2006) Clin. Cancer Res. 12:4916-4924). Despite these advantages, the relatively high dosages which are needed in order to obtain a pharmacological effect, the low encapsulation efficiencies due to the lack of an electrostatic attraction to the anionic nucleic acids and the poor cellular uptake represent major challenges in these two groups of lipid vehicles (Wang et al., (1987) Proc. Natl. Acad. Sci. 84:7851-7855 and Foged et al., (2006) International Journal of Pharmaceutics 331:160-166).
Compared to the anionic and neutral approaches, cationic liposomal carriers have a positive net surface charge, which facilitates rapid complex formation with negatively charged nucleic acids (Semple et al., (2001) Biochimica et Biophysica Acta 1510:152-166 and Leonetti et al., (2001) Cancer Gene Therapy 8:459-468). In addition, lipid complexes with a positive net charge are readily adsorbed onto the negatively charged cell membrane, leading to a high local nucleic acid concentration at the cell membrane, which supports their intracellular internalization. One example of such vectors is the polycationic liposomes designed by Santel and co-workers, which can mediate delivery of small interfering RNA (siRNA) molecules in endothelial cells in different mouse xenograft tumor models upon intravenous administration (Santel et al., (2006) Gene Therapy 16:1222-1234). Despite encouraging results, it has been observed that inhalable application of these polycationic liposomes evoked inflammation (Gutbier et al., (2010) Pulmonary Pharmacology & Therapeutics 23:334-344). Strong side effects, such as experimental animal death and induction of the immune system were also observed using other polycationic delivery approaches (Bitko et al., (2005) Nature Medicine 11.1:50-55). Although strong cell membrane attraction has advantages, such rapid and non-specific binding of cationic membranes to the anionic cells can also result in high toxicity levels. Aggregate formation with serum components and relatively short circulation lifetimes are additional hurdles to circumvent for the successful application of these carrier systems (Andreakos et al., (2009) Arthritis Rheum. 60:994-1005).
Another interesting strategy of cationic lipid assemblies is the pH sensitive cationic lipid particles of Tekmira pharmaceuticals. These lipid particles have been used successfully for the delivery of siRNAs into the liver and as demonstrated lately the functionality of these vectors depends on the ApoE protein and the use of LDL receptor (Semple et al., (2010) Nature Biotechnology 28:172-176 and Akinc et al., (2010) Molecular Therapy 18:1357-1364). Another example for efficient delivery of siRNA into the liver is the use of permanently charged cationic lipidoids as demonstrated in Akinc et al., (2008) Nature Biotechnology 28:561-569. However, the dependency of a liposomal delivery system to a specific natural protein or the restricted biodistribution, primarily liver accumulation in the case of cationic lipidoids, narrows the spectrum of in vivo applications.
Thus, the objective of this invention is to provide a method of preparing a drug delivery system, which can transport biologically active compounds, such as nucleic acids or small molecules, proteins and peptides, to the target cells. Another objective of this invention is to provide a mechanism of preparing a carrier, which could combine the advantages of the cationic and anionic liposomal delivery approaches, meaning high encapsulation efficiencies of drug and longer circulation times thus leading to improved tissue distribution and safety. The disclosure also provides compounds and compositions and the use thereof for improving in vitro and in vivo application of biologically active compounds.